The Study On The Promoting Effect Of Vascular Adventitial Activation On The Formation And Development Of Neointima In Transplant Vasculopathy

Transplant vasculopathy (TV) is a vascular proliferative disease. TV occurs in organ transplants and is the most significant obstacle to successful long-term survival. TV has many causative factors and pathologic manifestations in common with atherosclerosis and post-angioplasty restenosis (RS).Conventional understanding of the pathologic manifestations of TV includes migration and proliferation of smooth muscle cells (SMCs), infiltration with inflammatory cells, lipid aggregation in macrophages and SMCs, increased production of the extracellular matrix (ECM), and neointima formation. Accordingly, studies on vascular diseases have focused on the endothelia and smooth muscle dysfunction; less attention has been paid to the vascular adventitia.Arterial walls are heterogeneous three-layered structures composed of an intima, media, and adventitia. Each layer exhibits specific histological, biochemical, and functional characteristics, and, as such, each contributes in unique ways to maintaining vascular homeostasis and to regulating the vascular response to stress or injury. Compared with intima and media, the adventitia has been considered to be exclusively a supporting tissue that can provide nourishment to muscle layers. Recently, increasing attention has been paid to its role in a variety of vascular diseases, including AS and RS. Some studies have suggested that the vascular adventitia acts as an important regulator to modulate, directly or indirectly, the structure and function of the vascular wall. As the major cell type in the vascular adventitia, increasing studies showed that adventitial fibroblast was not a passive bystander but a active participant in response to diverse stimulations and thus contributed to neointima formation and vascular remodeling. Activation of the adventitial fibroblasts in response to cytokines, injury and stretch has been shown to stimulate phenotypic differentiation, migration, proliferation and synthesis of ECM. Well-known cytokines such as transforming growth factor beta (TGF-β1) and endothelin-1 (ET-1) can induce the transition of fibroblasts into myofibroblasts (MFs) which can traverse the ruptured media and participate in neointimal formation after balloon injury. Vascular adventitial fibroblasts can also secret a variety of cytokines, such as TGF-β1, monocyte chemoattrant protein-1 (MCP-1), interleukin (IL), ET-1, tumor necrosis factor alpha (TNFa), matrix metalloproteinases (MMPs) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Adventitia reactions are common in various vascular diseases. Porcine models showed that the inflammatory response after angioplasty occurred throughout the entire perivascular tissue before neointimal formation. A previous study from our research team showed that adventitial fibroblasts were activated in the early stage of atherosclerosis, and that the earliest expression of MCP-1 which is capable of regulating the transmigration of monocytes into the vessel wall occurred in the adventitial fibroblasts before the formation of intimal lesions in apoE knockout mice. It was reported that abundant a-smooth muscle actin (a-SMA) immunoreactive cells were present in the adventitia in porcine after balloon injury. Li et al. tracked the rat carotid adventitial fibroblasts which transduced withβ-galactosidase and convincingly demonstrated directly in vivo the ability of adventitial fibroblasts to contribute to neointimal growth by migrating into the neointima over time after balloon injury. In carotid artery-vein grafts, neointima formation was proceded by proliferation, differentiation into myofibroblasts and migration into neointima of adventitial fibroblasts. Neointima formation and/or atherosclerotic lesions have been observed in response to adventitial injury in various animal models. Recently, genge therapy has become a'hot spot'for preventing and treating cardiovascular diseases and the outer location of the adventitial fibroblast makes it suitable for drug delivery and gene therapy. Dourron et al. designed a novel replication-deficient adenovirus containing a fibroblast-active promoter that drives expression of the NADPH oxidase inhibitory sequence gp91ds (Ad-PDGFPR-gp91ds/eGFP). When this was delivered to the adventitia of the rat common carotid artery, it effectively reduced overall vascular 02·-and neointima formation. Though people carried out a lot of experiments and made some progress on the biological mechanism of vascular adventitia in vascular diseases, much mechanism especially in TV is still unclear. And there are few reports about it at present.Objectives:In the present study we constructed a successful allograft model in the rat via a cuff technique. In order to study the role of the activated vascular adventitia in the neointima formation and development in transplant vasculopathy, we observed the vascular adventitia especially the adventitial fibroblast phenotype differentiation, proliferation, migration and the mRNA and protein levels of various cytokines at the given time points after grafting both in vivo and in vitro. Furthermore, we investigated the inhibitive effect on the proliferation and migration of adventitial fibroblast by down-regulating the NADPH oxidase activity via RNA interference to silence the expression of p47phox, one subunit of NADPH oxidase. And the study will provide theory basis for gene therapy targeting at vascular adventitial fibroblast in preventing and treating the transplant vasculopathy. This study has been divided into three parts as following:Part I:Construction of a transplant vasculopathy animal model in the ratMethod:The procedure was carried out using an improved cuff technique. Thoracic aortas from SD rats transplanted into the abdominal aortas of Wistar rats worked as allografts, and isografts (SD to SD) were control. Grafts were removed on days 3,7, and 14 for HE staining and Weigert staining. The thicknesses of the neointima and media and intimal/medial thickness ratio were measured using a computer-assisted morphometry system (Image-Pro Plus 5.0).Result:After transplantation, the animal survival rate was about 90%and the patency rate was 100%. No detectable neointimal hyperplasia was found in 3-,7-and 14-day isografts as well as 3-day allografts. The neointima was initially found in 7-day allografts and significantly increased in 14-day allografts. The intimal thickness and intimal/medial thickness ratio were increased in 7-day allografts and peaked in 14-day allografts. Markedly reduced medial thickness in the process was not observed.Conclusion:We constructed a successful transplant vasculopathy animal model in the rat. It was characterized by simple operation, less cost and favorable repeatability. Thus, it can be used to study allograft pathologic mechanism.PartⅡ:The study on the role of the vascular adventitial activation in the neointima formation and development in transplant vasculopathy in vivoMethod:At given time points, allografts and isografts were removed. Paraffin-embedded sections were stained with immunohistochemistry to observe the changes in the expression of vimentin,α-SMA, Ki-67, NF-κB, MCP-1, ICAM-1, VCAM-1, MMP-7, TGF-β1, nitrotyrosine and p47phox in the vascular adventitia and intima/neointima. Fluorescence in situ hybridization (FISH) was performed to examine the expression of the mRNA for gp91phox. The mRNA levels of MCP-1, IL-1β, TNFα, ICAM-1, VCAM-1, MMP-7, TGF-β1, gp91phox and p47phox expressed in the grafts adventitia were quantified by quantitative real-time reverse transcriptase polymerase chain reaction (QRT-PCR) using SYBR Green technology.Result: 1. Vimentin-positive cells were mainly in the adventitia and intima or neointima in both isografts and allografts. a-SMA was expressed only in the media and was barely detectable in the adventitia or intima in isografts. In allografts, expression of a-SMA in the media was similar to that in isografts, but a-SMA-positive cells were first found mainly in adventitial fibroblasts after 3 days and gradually increased till 14 days. The neointima began to express a-SMA in 7-and 14-day allografts.2. In our experiment, no more than 4%of CD3-positive T cells were found in the adventitia in both isografts and allografts in any given time points after grafting, indicating that most of the proliferative cells were adventitial fibroblasts which might contribute to the neointima formation.3. Immunostaing for Ki-67 showed that in allografts, about 20%of adventitial fibroblasts and only a few intimal endothelial cells were proliferative after 3 days, and the number of proliferative cells was significantly increased in the adventitia and neointima after 7 and 14 days. Proliferative cells were barely detectable in all three layers in isografts. Surprisingly, there were neither obvious proliferative cells nor evident changes in the thickness in the media during the TV development.4. In the allografts, the expression of NF-κB, MCP-1, ICAM-1, VCAM-1, MMP-7, TGF-β1, nitrotyrosine and p47phox were first found in the adventitia and intima/neointima 3 days after grafting, and significantly increased in the adventitia and neointima after 7 and 14 days. In the isografts, only a few cells positive for NF-κB, MCP-1, ICAM-1, VCAM-1 and nitrotyrosine were found in the adventitia and positive cells were barely detectable in the intima or neointima.5.In the allografts, the expression of gp91phox mRNA were first found in the adventitia 3 days after grafting, and significantly increased after 7 and 14 days. Only a few positive cells were observed in the intima. However, the expression of gp91phox mRNA were barely detectable in the isografts.6. Compared with isografts, the mRNA levels of MCP-1, IL-1β, TNFα, ICAM-1, VCAM-1, MMP-7, TGF-β1, gp91phox and p47phox in the adventitia were increased 3 days after grafting, and peaked at 14 days.Conclusion: 1. In the early stage of TV, vascular adventitia especially the adventitial fibroblast was activated before the neointima formation. This process was characterized by the phenotypic differentiation into myofibroblast withα-SMA expression, the increased capacity of proliferation and synthesis of various active cytokines. Thus, we hypothesized that adventitial fibroblast might contribute to the neointima formation after its activation in the development of TV.2. Vascular adventitial inflammation is an early event in the neointima formation and development in TV.3. Oxidative stress occured through the development of TV might be one of the causes of the activation of vascular adventitia especially the adventitial fibroblast and be involved in the neointima formation and development in TV.PartⅢ:The study on the role of the adventitial fibroblasts in the neointima formation and development in transplant vasculopathy in vitroMethod:Allografts and isografts were removed 14 days after grafting. Adventitia was carefully stripped from isografts and allografts, and then cultured by the tissue explant method, respectively. Cells at passages 3 to 6 were used for further study. Immunofluorescence staining for vimentin, a-SMA and desmin was used to identify the cultured adventitial cells.The proliferation of cells was examined by CCK-8 kit and BrdU incorporation assay. PI staining by flow cytometry was used to examine the cell cycle of cultured cells. Cell migration assay was carried out using 24 well ThinCertTM cell culture inserts and scratch method. The mRNA levels of MCP-1, IL-1β, TNFα, ICAM-1, VCAM-1, MMP-7, TGF-β1, gp91phox and p47phox expressed in the cultured adventitial cells were quantified by reverse transcriptase polymerase chain reaction (RT-PCR) and the protein levels of MCP-1, ICAM-1, TGF-β1 and p47phox were quantified by Western blot. We designed and synthesized siRNAs targeting p47phox, one subunit of NADPH oxidase, and then screened one siRNA with high efficiency. p47phox siRNA was transfected into cultured adventitial fibroblasts from allografts to observe its inhibition effect on NADPH oxidase activity and cell proliferation and migration capacity.Result:1. Adventitia was cultured by the tissue explant method. About 4-7 days, small amounts of cells started to immigrate out of the explants and adhere to the flask bottom and for 10～14 days they reached 90%confluence. More than 90%cells from isografts are vimentin (+),desmin (-) andα-SMA (-), while about 90%cells from allografts are vimentin (+), desmin (-) andα-SMA(+).2. Compared with isografts, there is no significant difference between the two groups in the proliferation capacity of the cultured adventitial cells when cultured with 1%serum, but the proliferation capacity of the cultured adventitial cells from allografts were significantly increased when cultured with 5%or 10%serum. The same result was gained by the method of BrdU incorporation assay when cultured with 10%serum. PI staining by flow cytometry also showed the percentage of S and G2/M phase of the cultured adventitial cells from allografts was much higher than that from isografts.3. Both 24 well ThinCertTM cell culture inserts and scratch method demonstrated that the migration capacity of the cultured adventitial cells from allografts were significantly increased compared with that from isografts.4. Compared with isografts, the mRNA levels of MCP-1, IL-1β, TNFα, ICAM-1, VCAM-1, MMP-7, TGF-β1,gp91phox and p47phox in the cultured adventitial cells from allografts were significantly increased.5. Compared with isografts, the protein levels of MCP-1, ICAM-1, TGF-β1 and p47phox in the cultured adventitial cells from allografts were significantly increased.6. After 48 hours of the transfection of p47phox-siRNA-2#, the mRNA level of p47phox was effectively inhibited. Furthermore, the NADPH oxidase activity and the proliferation and migration capacity of the cultured adventitial cells from allografts were significantly decreased.Conclusion:1. About 90%cells from allografts differentiated into myofibroblasts while more than 90%cells from isografts were fibroblasts.2. Compared with isografts, the proliferation and migration capacity of the cultured adventitial cells from allografts were significantly increased.3. Compared with isografts, the synthesis and release of various active cytokines by the cultured adventitial cells from allografts were significantly increased.4. Oxidative stress was involved in the activation of adventitial fibroblasts, while activated adventitial fibroblasts might produce more superoxide anion and thus a positive feedback might be formed between them.5. p47phox siRNA effectively inhibited the proliferation and migration capacity of adventitial fibroblasts, and thus might be a new target for the gene therapy to attenuate the neointima formation in TV.